Development Of A Cytokine Chip

细胞因子具有多种调节功能，其中最重要的是调节T细胞分化。分泌的细胞因子谱已被证明可以预测不同的T细胞分化，特别是调节细胞介导性和体液免疫的细胞因子。为了在单个样本中同时测量不同的细胞因子谱，基于对感兴趣的分析物的固相免疫亲和提取，开发了一种基本的细胞因子芯片。重组链霉亲和素斑点阵列通过羰基双咪唑桥被衍生化到硅烷化玻璃载玻片表面。抗细胞因子抗体通过其碳水化合物部分被生物素化，并与Stetavidin结合。样品在分析前用激光染料标记，并与芯片孵化。在通过洗涤去除非反应性物质之后，在扫描激光密度计中读取结合分析物。在固定化化学和保持抗体分子活性的技术方面的改进使芯片对所有研究的50种细胞因子的灵敏度提高到1.0-2.5pg/ml。研究人员正在继续提高这一灵敏度水平，并进一步优化芯片读取机制。当使用细胞因子的模型混合物和人类临床样本时，该芯片显示出非常好的选择性和高度的特异性。该芯片目前正处于评估阶段，尚未用于分析重要的调节途径，包括造血、炎症、神经性调节、细胞调节和伤口愈合。 继续开发和改进一种静态抗体阵列，用于测量临床样本中的细胞因子和生长因子。自动芯片阅读器的获得极大地提高了抗体阵列的灵敏度和选择性。此外，双激光系统的使用扩大了阵列的用途。最近与美国国立卫生研究院临床中心危重护理中心建立的合作研究为抗体芯片开辟了新的临床应用。目前，一组50-100种特异性抗体正在接受免疫化学评估，以纳入患者筛查芯片。该芯片将用于筛选患有炎症性肺病的儿童患者，以寻找调节性细胞因子、疼痛相关神经肽和其他与呼吸系统疾病免疫功能相关的调节剂。

Cytokines serve a variety of regulatory functions, one of the most important being the regulation of T-cell differentiation. Secreted cytokine profiles have been demonstrated to be predictive of different T-cell differentiation, especially cytokines regulating cell-mediated and humoral immunity. In order to measure different cytokine profiles simultaneously in individual samples, a basic cytokine chip has been developed based on solid-phase immunoaffinity extraction of the analytes of interest. Arrays of recombinant streptavidin spots were derivatized to the surfaces of silanized glass microscope slides via a carbonyldiimidazole bridge. Anti-cytokine antibodies were biotinylated via their carbohydrate moieties and bound to the steptavidin. Samples were labeled with a laser dye prior to analysis and incubated with the chip. Following removal of non-reactive materials by washing, the bound analytes were read in a scanning laser densitometer. Improvements in both the immobilization chemistry and techniques for keeping the antibody molecules active have increased the sensitivity of the chip to 1.0-2.5 pg/ml for all 50 cytokines studied. Studies are continuing to improve this level of sensitivity as well as further optimize the chip reading mechanism. The chip exhibits very good selectivity and a high degree of specificity when using both model mixtures of cytokines and human clinical samples. The chip is now in the evaluation stage prior to its use in such applications as the analysis of important regulatory pathways, including hematopoesis, inflammation neurogenic regulation, cellular regulation, and wound healing. Work has continued to develop and improve a static antibody array for measuring cytokines and growth factors in clinical samples. The acquisition of an automated chip reader greatly improved both sensitivity and selectivity of the antibody arrays. Additionally, the use of a dual laser system has expanded the usefulness of the array. Collaborative studies recently established with the Critical Care Center of the NIH Clinical Center have opened up new clinical applications for the antibody chip. At present, a panel of 50-100 specific antibodies is being immunochemically evaluated for inclusion in a patient-screening chip. This chip will be used to screen pediatric patients with inflammatory lung disease for regulatory cytokines, pain-associated neuropeptides, and other modulators associated with immune function in respiratory disease.